Human lysyl-tRNA synthetase phosphorylation promotes HIV-1 proviral DNA transcription.

Human lysyl-tRNA synthetase (LysRS) was previously shown to be re-localized from its normal cytoplasmic location in a multi-aminoacyl-tRNA synthetase complex (MSC) to the nucleus of HIV-1 infected cells. Nuclear localization depends on S207 phosphorylation but the nuclear function of pS207-LysRS in the HIV-1 lifecycle is unknown. Here, we show that HIV-1 replication was severely reduced in a S207A-LysRS knock-in cell line generated by CRISPR/Cas9; this effect was rescued by S207D-LysRS. LysRS phosphorylation up-regulated HIV-1 transcription, as did direct transfection of Ap4A, an upstream transcription factor 2 (USF2) activator that is synthesized by pS207-LysRS. Overexpressing an MSC-derived peptide known to stabilize LysRS MSC binding inhibited HIV-1 replication. Transcription of HIV-1 proviral DNA and other USF2 target genes was reduced in peptide-expressing cells. We propose that nuclear pS207-LysRS generates Ap4A, leading to activation of HIV-1 transcription. Our results suggest a new role for nuclear LysRS in facilitating HIV-1 replication and new avenues for antiviral therapy.

Antibody Deficiency in Patients with Biallelic KARS1 Mutations.

Biallelic KARS1 mutations cause KARS-related diseases, a rare syndromic condition encompassing central and peripheral nervous system impairment, heart and liver disease, and deafness. KARS1 encodes the t-RNA synthase of lysine, an aminoacyl-tRNA synthetase, involved in different physiological mechanisms (such as angiogenesis, post-translational modifications, translation initiation, autophagy and mitochondrial function). Although patients with immune-hematological abnormalities have been individually described, results have not been collectively discussed and functional studies investigating how KARS1 mutations affect B cells have not been performed. Here, we describe one patient with severe developmental delay, sensoneurinal deafness, acute disseminated encephalomyelitis, hypogammaglobulinemia and recurrent infections. Pathogenic biallelic KARS1 variants (Phe291Val/ Pro499Leu) were associated with impaired B cell metabolism (decreased mitochondrial numbers and activity). All published cases of KARS-related diseases were identified. The corresponding authors and researchers involved in the diagnosis of inborn errors of immunity or genetic syndromes were contacted to obtain up-to-date clinical and immunological information. Seventeen patients with KARS-related diseases were identified. Recurrent/severe infections (9/17) and B cell abnormalities (either B cell lymphopenia [3/9], hypogammaglobulinemia [either IgG, IgA or IgM; 6/15] or impaired vaccine responses [4/7]) were frequently reported. Immunoglobulin replacement therapy was given in five patients. Full immunological assessment is warranted in these patients, who may require detailed investigation and specific supportive treatment.

MRGPRX2 signaling involves the Lysyl-tRNA synthetase and MITF pathway.

MRGPRX2, a G-protein-coupled-seven transmembrane domain receptor, is mainly expressed in mast cells and neurons and is involved in skin immunity and pain. It is implicated in the pathophysiology of non-IgE-mediated immediate hypersensitivity and has been related to adverse drug reactions. Moreover, a role has been proposed in asthma, atopic dermatitis, contact dermatitis, and chronic spontaneous urticaria. Although it has a prominent role in disease, its signaling transduction is poorly understood. This study shows that MRGPRX2 activation with substance P increased Lysyl t-RNA synthetase (LysRS) translocation to the nucleus. LysRS is a moonlighting protein with a dual role in protein translation and IgE signaling in mast cells. Upon allergen- IgE-FcεRI crosslinking, LysRS is translocated to the nucleus and activates microphthalmia-associated transcription factor (MITF) activity. In this study, we found that MRGPRX2 triggering led to MITF phosphorylation and increased MITF activity. Therefore, overexpression of LysRS increased MITF activity after MRGPRX2 activation. MITF silencing reduced MRGPRX2-dependent calcium influx and mast cell degranulation. Furthermore, a MITF pathway inhibitor, ML329, impaired MITF expression, calcium influx, and mast cell degranulation. Moreover, drugs such as atracurium, vancomycin, and morphine, reported to induce MRGPRX2-dependent degranulation, increased MITF activity. Altogether, our data show that MRGPRX2 signaling enhances MITF activity, and its abrogation by silencing or inhibition resulted in defective MRGPRX2 degranulation. We conclude that MRGPRX2 signaling involves the LysRS and MITF pathway. Thus, MITF and MITF-dependent targets may be considered therapeutic approaches to treat pathologies where MRGPRX2 is implicated.

Toolkit of Approaches To Support Target-Focused Drug Discovery for Plasmodium falciparum Lysyl tRNA Synthetase.

There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.

Changes in subcellular localization of Lysyl-tRNA synthetase and the 67-kDa laminin receptor in epithelial ovarian cancer metastases.

BACKGROUND: Although lysyl-tRNA synthetase (KARS1) is predominantly located in the cytosol, it is also present in the plasma membrane where it stabilizes the 67-kDa laminin receptor (67LR). This physical interaction is strongly increased under metastatic conditions. However, the dynamic interaction of these two proteins and the turnover of KARS1 in the plasma membrane has not previously been investigated. OBJECTIVE: Our objective in this study was to identify the membranous location of KARS1 and 67LR and investigate if this changes with the developmental stage of epithelial ovarian cancer (EOC) and treatment with the inhibitor BC-K01. In addition, we evaluated the therapeutic efficacy of BC-K01 in combination with paclitaxel, as the latter is frequently used to treat patients with EOC. METHODS: Overall survival and prognostic significance were determined in EOC patients according to KARS1 and 67LR expression levels as determined by immunohistochemistry. Changes in the location and expression of KARS1 and 67LR were investigated in vitro after BC-K01 treatment. The effects of this compound on tumor growth and apoptosis were evaluated both in vitro and in vivo. RESULTS: EOC patients with high KARS1 and high 67LR expression had lower progression-free survival rates than those with low expression levels of these two markers. BC-K01 reduced cell viability and increased apoptosis in combination with paclitaxel in EOC cell xenograft mouse models. BC-K01 decreased membranous KARS1 expression, causing a reduction in 67LR membrane expression in EOC cell lines. BC-K01 significantly decreased in vivo tumor weight and number of nodules, especially when used in combination with paclitaxel. CONCLUSIONS: Co-localization of KARS1 and 67LR in the plasma membrane contributes to EOC progression. Inhibition of the KARS1-67LR interaction by BC-K01 suppresses metastasis in EOC.

Human lysyl-tRNA synthetase evolves a dynamic structure that can be stabilized by forming complex.

The evolutionary necessity of aminoacyl-tRNA synthetases being associated into complex is unknown. Human lysyl-tRNA synthetase (LysRS) is one component of the multi-tRNA synthetase complex (MSC), which is not only critical for protein translation but also involved in multiple cellular pathways such as immune response, cell migration, etc. Here, combined with crystallography, CRISPR/Cas9-based genome editing, biochemistry, and cell biology analyses, we show that the structures of LysRSs from metazoan are more dynamic than those from single-celled organisms. Without the presence of MSC scaffold proteins, such as aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2), human LysRS is free from the MSC. The interaction with AIMP2 stabilizes the closed conformation of LysRS, thereby protects the essential aminoacylation activity under stressed conditions. Deleting AIMP2 from the human embryonic kidney 293 cells leads to retardation in cell growth in nutrient deficient mediums. Together, these results suggest that the evolutionary emergence of the MSC in metazoan might be to protect the aminoacyl-tRNA synthetase components from being modified or recruited for use in other cellular pathways.

Ketogenic Diet for KARS-Related Mitochondrial Dysfunction and Progressive Leukodystrophy.

KARS encodes lysyl-tRNA synthetase, which is essential for protein translation. KARS mutations sometimes cause impairment of cytoplasmic and mitochondrial protein synthesis, and sometimes lead to progressive leukodystrophies with mitochondrial signature and psychomotor regression, and follow a rapid regressive course to premature death. There has been no disease-modifying therapy beyond supportive treatment. We present a 5-year-old male patient with an asymmetrical leukodystrophy who showed overt evidence of mitochondrial dysfunction, including elevation of lactate on brain MR spectroscopy and low oxygen consumption rate in fibroblasts. We diagnosed this patient's condition as KARS-related leukodystrophy with cerebral calcification, congenital deafness, and evidence of mitochondrial dysfunction. We employed a ketogenic diet as well as multiple vitamin supplementation with the intention to alleviate mitochondrial dysfunction. The patient showed alleviation of his psychomotor regression and even partial restoration of his abilities within 4 months. This is an early report of a potential disease-modifying therapy for KARS-related progressive leukodystrophy without appreciable adverse effects.

Structural analyses of the malaria parasite aminoacyl-tRNA synthetases provide new avenues for antimalarial drug discovery.

Malaria is a parasitic illness caused by the genus Plasmodium from the apicomplexan phylum. Five plasmodial species of P. falciparum (Pf), P. knowlesi, P. malariae, P. ovale, and P. vivax (Pv) are responsible for causing malaria in humans. According to the World Malaria Report 2020, there were 229 million cases and ~ 0.04 million deaths of which 67% were in children below 5 years of age. While more than 3 billion people are at risk of malaria infection globally, antimalarial drugs are their only option for treatment. Antimalarial drug resistance keeps arising periodically and thus threatens the main line of malaria treatment, emphasizing the need to find new alternatives. The availability of whole genomes of P. falciparum and P. vivax has allowed targeting their unexplored plasmodial enzymes for inhibitor development with a focus on multistage targets that are crucial for parasite viability in both the blood and liver stages. Over the past decades, aminoacyl-tRNA synthetases (aaRSs) have been explored as anti-bacterial and anti-fungal drug targets, and more recently (since 2009) aaRSs are also the focus of antimalarial drug targeting. Here, we dissect the structure-based knowledge of the most advanced three aaRSs-lysyl- (KRS), prolyl- (PRS), and phenylalanyl- (FRS) synthetases in terms of development of antimalarial drugs. These examples showcase the promising potential of this family of enzymes to provide druggable targets that stall protein synthesis upon inhibition and thereby kill malaria parasites selectively.

Immune recognition of lysyl-tRNA synthetase and isoleucyl-tRNA synthetase by anti-OJ antibody-positive sera.

OBJECTIVE: Anti-aminoacyl-tRNA synthetase (anti-ARS) antibodies are useful for identifying a clinical subset of patients with idiopathic inflammatory myopathies (IIMs). Anti-OJ antibodies, which recognize multi-enzyme synthetase complexes including isoleucyl-tRNA synthetase (IARS) and lysyl-tRNA synthetase (KARS), are among the anti-ARS antibodies. Although testing antibodies to other ARSs have been used clinically, no validated immunoassays for detecting anti-OJ antibodies are available. We aimed to establish an anti-OJ ELISA. METHODS: Serum samples were collected from 279 patients with IIMs and 22 patients with idiopathic interstitial pneumonia. Sixty-four of the samples that had been confirmed to be negative for anti-OJ by standard immunoprecipitation were used as the negative control, and 12 anti-OJ-positive reference sera were used as the positive control. Antibodies to IARS and KARS were assayed by ELISA using biotinylated recombinant proteins generated by in vitro transcription/translation. RESULTS: The anti-OJ-positive sera strongly reacted with the KARS and IARS recombinant proteins in ELISA. Although all 12 reference sera were positive in the anti-KARS ELISA, 4 of the 64 anti-OJ-negative sera were also weakly positive. The sensitivity and the specificity were 100% and 93.8%, respectively. Since our anti-KARS ELISA performed well, showing a high agreement with the results for immunoprecipitation (Cohen's κ > 0.8), the remaining 237 samples were also tested. Thirteen anti-KARS-positive sera were newly found by ELISA, all of which were anti-OJ positive by immunoprecipitation. CONCLUSION: Immunoassays for detecting anti-OJ antibodies using KARS and IARS recombinant proteins were developed. Our ELISAs performed well, with very high agreement of the results by immunoprecipitation and can be applied to the first reliable, easy-to-use measurement assays for anti-OJ antibodies.

Discovery of novel potent migrastatic Thiazolo[5,4-b]pyridines targeting Lysyl-tRNA synthetase (KRS) for treatment of Cancer metastasis.

Recently, non-canonical roles of Lysyl-tRNA Synthetase (KRS), which is associated with cell migration and cancer metastasis, have been reported. Therefore, KRS has emerged as a promising target for the treatment of cell migration-related diseases, especially cancer metastasis, although the satisfying chemical inhibitors targeting KRS have not yet been identified. Here, we report the discovery of novel, mechanistically unique, and potent cell migration inhibitors targeting KRS, including the chemical and biological studies on the most effective N,N-dialkylthiazolo [5,4-b]pyridin-2-amine (SL-1910). SL-1910 exhibited highly potent migration inhibition (EC50 = 81 nM against the mutant KRS-overexpressed MDA-MB-231 cells) and was superior to the previously reported KRS inhibitor (migration inhibitory EC50 = 8.5 µM against H226 cells). The KRS protein binding study via fluorescence-based binding titration and KRS protein 2D-NMR mapping study, in vitro concentration-dependent cell migration inhibition, and in vivo anti-metastatic activity of SL-1910, which consists of a new scaffold, have been reported in this study. In addition, in vitro absorption, distribution, metabolism, and excretion studies and mouse pharmacokinetics experiments for SL-1910 were conducted.

Structural analyses of a human lysyl-tRNA synthetase mutant associated with autosomal recessive nonsyndromic hearing impairment.

Aminoacyl-tRNA synthetases (AARSs) catalyze the ligation of amino acids to their cognate tRNAs and therefore play an essential role in protein biosynthesis in all living cells. The KARS gene in human encodes both cytosolic and mitochondrial lysyl-tRNA synthetase (LysRS). A recent study identified a missense mutation in KARS gene (c.517T > C) that caused autosomal recessive nonsyndromic hearing loss. This mutation led to a tyrosine to histidine (YH) substitution in both cytosolic and mitochondrial LysRS proteins, and decreased their aminoacylation activity to different levels. Here, we report the crystal structure of LysRS YH mutant at a resolution of 2.5 Å. We found that the mutation did not interfere with the active center, nor did it cause any significant conformational changes in the protein. The loops involved in tetramer interface and tRNA anticodon binding site showed relatively bigger variations between the mutant and wild type proteins. Considering the differences between the cytosolic and mitochondrial tRNAlyss, we suggest that the mutation triggered subtle changes in the tRNA anticodon binding region, and the interferences were further amplified by the different D and T loops in mitochondrial tRNAlys, and led to a complete loss of the aminoacylation of mitochondrial tRNAlys.

Synthetic post-translational modifications of elongation factor P using the ligase EpmA.

Canonically, tRNA synthetases charge tRNA. However, the lysyl-tRNA synthetase paralog EpmA catalyzes the attachment of (R)-ß-lysine to the ε-amino group of lysine 34 of the translation elongation factor P (EF-P) in Escherichia coli. This modification is essential for EF-P-mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post-translational modification of K34 in EF-P with eight noncanonical substrates. In addition, acetylated EF-P was generated using an amber suppression system. The impact of these synthetically modified EF-P variants on in vitro translation of a polyproline-containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)-ß-lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF-P, but also opens a new route to post-translationally modify proteins using EpmA.

Atomic Resolution Analyses of Isocoumarin Derivatives for Inhibition of Lysyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases, the essential enzyme family for protein translation, are attractive targets for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The antimalarial natural product cladosporin was discovered recently as a novel lysyl-tRNA synthetase (LysRS) specific inhibitor. Here, we report a thorough analysis of cladosporin derivatives using chemical synthesis, biophysical, and biochemical experiments. A series of isocoumarin derivatives with only one nonhydrogen atom/bond change per compound was synthesized. These changes include replacements of methyltetrahydropyran moiety by methylcyclohexane or cyclohexane, lactone by lactam, hydroxyl groups by methoxyl groups, and dismission of the chiral center at C3 with a Δ3,4 double bond. We evaluated these compounds by thermal shift assays and enzymatic experiments and further studied their molecular recognition by the Plasmodium falciparum LysRS through total five high-resolution crystal structures. Our results showed that the methyltetrahydropyran moiety of cladosporin could be replaced by a more stable methylcyclohexane without reducing binding ability. Removing the methyl group from the methylcyclohexane moiety slightly decreased the interaction with LysRS. Besides, the replacement with a lactam group or a conjugated Δ3,4 double bond within the scaffold could be two more options to optimize the compound. Lastly, the two phenolic hydroxyl groups were critical for the compounds to bind LysRS. The detailed analyses at atomic resolution in this study provide a foundation for the further development of new antibiotics from cladosporin derivatives.

Stabilization of Intrinsically Disordered DKK2 Protein by Fusion to RNA-Binding Domain.

Intrinsic disorders are a common feature of hub proteins in eukaryotic interactomes controlling the signaling pathways. The intrinsically disordered proteins (IDPs) are prone to misfolding, and maintaining their functional stability remains a major challenge in validating their therapeutic potentials. Considering that IDPs are highly enriched in RNA-binding proteins (RBPs), here we reasoned and confirmed that IDPs could be stabilized by fusion to RBPs. Dickkopf2 (DKK2), Wnt antagonist and a prototype IDP, was fused with lysyl-tRNA synthetase (LysRS), with or without the fragment crystallizable (Fc) domain of an immunoglobulin and expressed predominantly as a soluble form from a bacterial host. The functional competence was confirmed by in vitro Wnt signaling reporter and tube formation in human umbilical vein endothelial cells (HUVECs) and in vivo Matrigel plug assay. The removal of LysRS by site-specific protease cleavage prompted the insoluble aggregation, confirming that the linkage to RBP chaperones the functional competence of IDPs. While addressing to DKK2 as a key modulator for cancer and ischemic vascular diseases, our results suggest the use of RBPs as stabilizers of disordered proteinaceous materials for acquiring and maintaining the structural stability and functional competence, which would impact the druggability of a variety of IDPs from human proteome.

Mutations in KARS cause a severe neurological and neurosensory disease with optic neuropathy.

Mutations in genes encoding aminoacyl-tRNA synthetases have been reported in several neurological disorders. KARS is a dual localized lysyl-tRNA synthetase and its cytosolic isoform belongs to the multiple aminoacyl-tRNA synthetase complex (MSC). Biallelic mutations in the KARS gene were described in a wide phenotypic spectrum ranging from nonsyndromic deafness to complex impairments. Here, we report on a patient with severe neurological and neurosensory disease investigated by whole-exome sequencing and found to carry biallelic mutations c.683C>T (p.Pro228Leu) and c.871T>G (p.Phe291Val), the second one being novel, in the KARS gene. The patient presented with an atypical clinical presentation with an optic neuropathy not previously reported. At the cellular level, we show that cytoplasmic KARS was expressed at a lower level in patient cells and displayed decreased interaction with MSC. In vitro, these two KARS variants have a decreased aminoacylation activity compared with wild-type KARS, the p.Pro228Leu being the most affected. Our data suggest that dysfunction of cytoplasmic KARS resulted in a decreased level of translation of the nuclear-encoded lysine-rich proteins belonging to the respiratory chain complex, thus impairing mitochondria functions.

Side chain rotameric changes and backbone dynamics enable specific cladosporin binding in Plasmodium falciparum lysyl-tRNA synthetase.

Cladosporin (CLD) is a fungal metabolite that kills the malaria parasite via inhibiting its cytoplasmic lysyl-tRNA synthetase (KRS) and abrogating protein translation. Here we provide structural and drug selectivity analyses on CLD interacting residues in apo and holo KRSs from Plasmodium falciparum, Homo sapiens, Cryptosporidium parvum, and Mycobacterium ulcerans. We show that both gross and subtle alterations in protein backbone and sidechains drive the active site structural plasticity that allows integration of CLD in KRSs. The ligand-induced fit of CLD in PfKRS is marked by closure and stabilization of three disordered loops and one alpha helix. However, these structural rearragements are not evident in KRS-CLD complexes from H. sapiens, C. parvum, or M. ulcerans. Strikingly, CLD fits into the MuKRS active site due to a remarkable rotameric alteration in its clash-prone methionine residue that provides accommodation for the methyl moiety in CLD. Although the high concentrations of drugs used for Hs, Cp, and MuKRS-CLD complexes in co-crystallization studies enable elucidation of a structural framework for understanding drug binding in KRSs, we propose that these data should be concurrently assessed via biochemical studies of potency and drug selectivity given the poor cell-based activity of CLD against human and bacterial cells. Our comprehensive analyses of KRS-CLD interactions, therefore, highlight vital issues in structure-based drug discovery studies.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Nucleic Acid-Dependent Structural Transition of the Intrinsically Disordered N-Terminal Appended Domain of Human Lysyl-tRNA Synthetase.

Eukaryotic lysyl-tRNA synthetases (LysRS) have an N-terminal appended tRNA-interaction domain (RID) that is absent in their prokaryotic counterparts. This domain is intrinsically disordered and lacks stable structures. The disorder-to-order transition is induced by tRNA binding and has implications on folding and subsequent assembly into multi-tRNA synthetase complexes. Here, we expressed and purified RID from human LysRS (hRID) in Escherichia coli and performed a detailed mutagenesis of the appended domain. hRID was co-purified with nucleic acids during Ni-affinity purification, and cumulative mutations on critical amino acid residues abolished RNA binding. Furthermore, we identified a structural ensemble between disordered and helical structures in non-RNA-binding mutants and an equilibrium shift for wild-type into the helical conformation upon RNA binding. Since mutations that disrupted RNA binding led to an increase in non-functional soluble aggregates, a stabilized RNA-mediated structural transition of the N-terminal appended domain may have implications on the functional organization of human LysRS and multi-tRNA synthetase complexes in vivo.

Genetic Code Expansion Method for Temporal Labeling of Endogenously Expressed Proteins.

We here present a method that combines genetic code expansion with CRISPR/Cas9 genome engineering to label endogenously expressed proteins with high spatiotemporal resolution. The method exploits the use of an orthogonal tRNA/tRNA synthetase pair in conjugation with noncanonical amino acids to create stop codon read through events. To demonstrate the functionality of the method, we pulse labeled endogenous ß-actin and tumor protein p53 with a minimally invasive HA tag at their C-termini. Targeting the protein label with a proximity ligation assay plus real time imaging facilitates seamless quantification of the protein synthesis rate and spatial localization at the single cell level. The presented approach does not interfere with any physiological control of cellular expression, nor did we observe any perturbation of endogenous protein functions.

Lysyl-Transfer RNA Synthetase Induces the Maturation of Dendritic Cells through MAPK and NF-κB Pathways, Strongly Contributing to Enhanced Th1 Cell Responses.

In addition to essential roles in protein synthesis, lysyl-tRNA synthetase (KRS) is secreted to trigger a proinflammatory function that induces macrophage activation and TNF-α secretion. KRS has been associated with autoimmune diseases such as polymyositis and dermatomyositis. In this study, we investigated the immunomodulatory effects of KRS on bone marrow-derived dendritic cells (DCs) of C57BL/6 mice and subsequent polarization of Th cells and analyzed the underlying mechanisms. KRS-treated DCs increased the expression of cell surface molecules and proinflammatory cytokines associated with DC maturation and activation. Especially, KRS treatment significantly increased production of IL-12, a Th1-polarizing cytokine, in DCs. KRS triggered the nuclear translocation of the NF-κB p65 subunit along with the degradation of IκB proteins and the phosphorylation of MAPKs in DCs. Additionally, JNK, p38, and ERK inhibitors markedly recovered the degradation of IκB proteins, suggesting the involvement of MAPKs as the upstream regulators of NF-κB in the KRS-induced DC maturation and activation. Importantly, KRS-treated DCs strongly increased the differentiation of Th1 cells when cocultured with CD4+ T cells. The addition of anti-IL-12-neutralizing Ab abolished the secretion of IFN-γ in the coculture, indicating that KRS induces Th1 cell response via DC-derived IL-12. Moreover, KRS enhanced the OVA-specific Th1 cell polarization in vivo following the adoptive transfer of OVA-pulsed DCs. Taken together, these results indicated that KRS effectively induced the maturation and activation of DCs through MAPKs/NF-κB-signaling pathways and favored DC-mediated Th1 cell response.